Objective: We investigated the diurnal variation in oxygen consumption to determine the optimal time periods of calorimetry in preterm infants. Methods: Oxygen consumption (VO2) was measured continuously for 24 h using indirect calorimetry. Twenty-two premature infants with gestational age of 27–31 (31±1.7) weeks were enrolled in the study. Heart rate, respiratory rate, oxygen saturation, skin and rectal temperature and physical activity were monitored continuously. Results: The averaged values of VO2 showed a significant pattern (P&#60;0.0001) of circadian rhythm with a peak in the afternoon and a nadir during the night with significantly differences between the mean VO2 values. A circadian variation of VO2 was found in more than 80% of preterm infants and was unrelated to gestational age, physical activity or environmental stress. The infants spent 90.4% of their time sleeping. Conclusions: These findings indicate the possible existence of an endogenous circadian rhythm of VO2 in preterm infants beginning shortly after birth. Day-night variations of VO2 should be considered when VO2 values are extrapolated from short measurement periods to prevent overestimation of values.